Project Summary Dynamic protein phosphorylation regulates virtually all biological processes. Selective dephosphorylation of specific substrates or specific sites on a given substrate is critical for key cellular events. Our recent studies have uncovered an unusual example of site-specific dephosphorylation by the multi-subunit Striatin-interacting phosphatase and kinase complex called STRIPAK, which controls Hippo pathway activation. The Hippo pathway is critical for tissue homeostasis and tumor suppression in multicellular organisms. Dysregulation of this pathway drives tumor formation in flies, mice, and humans. Through cell-surface receptors and cytoskeletal complexes (e.g. NF2), extracellular signals, including cell-cell contact, activate a core kinase cascade formed by the Hippo kinases MST1/2, the NDR family kinases LATS1/2, and the adaptor proteins SAV1 and MOB1. Activated LATS1/2 phosphorylate and prevent the nuclear accumulation of the transcription factors YAP/TAZ. When the Hippo pathway is off, YAP/TAZ translocate to the nucleus and form functional hybrid transcriptional factors with TEADs to promote the transcription of proliferative and pro-survival genes. Activation of MST1/2 initiates Hippo signaling and requires trans-autophosphorylation in the T-loop (MST2 T180). We have recently shown that the PP2A complex STRIPAK blocks MST2 activation through feedback inhibition. Active MST2 auto-phosphorylates multiple sites in the linker. The adaptor protein SLMAP in STRIPAK binds the phospho-linker and promotes MST2 pT180 dephosphorylation, thus ensuring low steady-state MST2 activation. SAV1 promotes MST2 activation by suppressing STRIPAKSLMAP-mediated dephosphorylation of MST2 pT180. The physiological function, regulation, and mechanism of action of STRIPAKSLMAP remain unresolved. We hypothesize that STRIPAKSLMAP integrates upstream inputs through SAV1 to control MST1/2 activation. Because STRIPAKSLMAP contains both a kinase and a phosphatase, the delicate balance between the opposite catalytic activities of the two enzymes in the same complex may play a critical role in toggling the activation status of Hippo signaling. Thus, understanding the architecture, assembly mechanism, and regulation of STRIPAKSLMAP is crucial to the eventual understanding of how the core Hippo pathway is regulated by upstream signals. This proposal aims to determine the molecular mechanism and structural basis of this opposing regulation of the Hippo kinases MST1/2 by SAV1 and STRIPAKSLMAP, and to dissect the architecture, assembly mechanism, and regulation of STRIPAKSLMAP in the context of Hippo signaling. This research will significantly advance our fundamental understanding of the regulation of the Hippo signaling network and may uncover novel ways of exploiting defects in the Hippo pathway to treat human diseases.